Hepatoprotectent phenylbutyratyrate drug conjugates, and their compositions preparation and methods of use thereof

ABSTRACT

In this current invention our primary aim to provide a phenylbutyrate conjugates and composition suitable for oral, or parenteral administration which overcomes the abovementioned problems. Phenylbutyrate conjugates are provided, which have a phenylbutyrate moiety covalently linked to a second drug such as Acetaminophen, or other drug molecules which induced hepatotoxicity. A compound for use in the treatment or prevention of a disease selected from hepatotoxicity, drug induced hepatotoxicity, or liver necrosis, and nonalcoholic steatohepatitis (NASH), and treat Pain, fever, cancer, inflammation, a composition for the treatment of ischemic injury or nerve damage in a human or animal, the compound having a structure selected from: **F-I** Formula and Formula **F-II* or anhydride, or pharmaceutically acceptable salt solvate thereof. It is one or more compounds or pharmaceutically acceptable salt or composition comprising a solvate of the foregoing.

FIELD OF INVENTION

The present invention relates to the discovery that 4-Phenylbutyricacid(PBA), Phenyl butyricacid esters and their pharmaceutically acceptablesalts, when co-administered in effective amounts with a drug or otherbioactive agent which typically (in the absence of the PBA compound)produces significant hepatotoxicity as a secondary indication, willsubstantially reduce or even eliminate such drug induced hepatotoxicityand liver necrosis and chronic alcohol consumption induced alcoholicliver steatosis, cirrhosis, and hepatocellular carcinoma. Favorabletherapeutic intervention results from the use of the present inventionhaving the effect of reducing hepatotoxicity associated with theadministration of certain drugs and other bioactive agents and incertain instance of allowing the administration of higher dose of acompound which, without co administration would produce hepatotoxicitywhich limits or even negates the therapeutic value of the compound.

BACKGROUND

Phenylbutyrate (PBA) is an aromatic short-chain fatty acid which is achemical derivative of butyric acid naturally produced by colonicbacteria fermentation. At the intestinal level butyrate exerts amultitude of activities such as diminish colonel cancer, improvement ofmucosal inflammation, and improvement in oxidative status. Similarly,phenylbutyrate displays potentially favorable effects on manypathologies including cancer, genetic metabolic syndromes, neuropathies,diabetes, hemoglobinopathies, and the chemical chaperone—PBA is a drugapproved by the U.S. Food and Drug Administration for urea cycledisorders treatment. The mechanisms by which PBA exerts these effectsare different. Some of them are connected with the regulation of geneexpression, playing the role of a histone deacetylase inhibitor, whileothers contribute to the ability of rescuing conformationalabnormalities of proteins, serving as chemical chaperone, and some arededicated to its metabolic characteristic enabling excretion of toxicammonia, thus acting as ammonia scavenger. Phenylbutyrate may exertvariable effects depending on the cell type, thus the term “butyrateparadox” has been proposed. These data indicate a broad spectrum ofbeneficial effects evoked by PBA with a high potential in therapy,unfolded protein response related proteins including GRP78, GRP94, C/EBPhomologous protein, phospho-eIF-2α, eIF-2α, phospho-JNK1 (p46) andphospho-JNK2/3 (p54), JNK1, IRE-1α, PERK, and sXBP-1. Endoplasmicreticulum (ER) stress is closely connected to autophagy. When cells areexposed to ER stress, cells exhibit enhanced protein degradation andform autophagosomes. From the past decade numerous studies have shownthat endoplasmic reticulum (ER) stress contributes to the progression ofliver disease (Ji and Kaplowitz, 2003; Malhi and Kaufman, 2011; Tan etal., 2013and Dara et al., 2011; Fernandez et al., 2013). ER stress andinitiation of the unfolded protein response (UPR) is caused byaccumulation of unfolded proteins in the ER, a cellular organelle thatis important for the regulation of calcium homeostasis, lipidmetabolism, and protein synthesis. The UPR pathway includes induction ofseveral molecular chaperones that restore cellular homeostasis bypromoting the folding or degradation of unfolded proteins; Thatalleviates ER stress by assisting in protein folding (Roy et al., 2015).Recently Kusuma et al reported PBA prevents murine dietarysteatohepatitis caused by trans-fatty acids plus fructose by minimizingER stress (Morinaga et al., 2015). This invention suggests that 4-PBAconjugates are potential therapeutic agents against ER stress-associatedpathologic situations; drug induced hepatotoxicity and liver necrosisand chronic alcohol consumption induced alcoholic liver steatosis,cirrhosis, and hepatocellular carcinoma.

However, most widely used drug Acetaminophen (APAP) also known asparacetamol, N-(4-hydroxyphenyl)acetamide, orN-(4-hydroxyphenyl)ethanamide), is an effective and safeanalgesic/antipyretic drug that is used around the world [1].Acetaminophen is the analgesic that is widely used for the treatment ofthe various conditions associated with pain and fever. For example,acetaminophen is used to manage post-operative pain or trauma, andosteoarthritis, pain caused by chronic inflammatory conditions such asrheumatoid arthritis and lower back pain. Optionally, acetaminophen isused to treat pain due to nociceptive/neuropathic mixed etiologies, suchas cancer or fibromyalgia. The administration of excessive Acetaminophen(APAP) induces Acetaminophen (APAP) hepatotoxicity is the most commoncause of death due to acute liver failure in the developed world and isincreasingly recognized as a significant public health problem (1, 2).The initial event in APAP induced hepatotoxicity is a toxic-metabolicinjury leading to hepatocyte death by necrosis and apoptosis whichrapidly progresses to acute liver failure. Acetaminophen (APAP) overdoseinduces severe oxidative stress followed by hepatocyteapoptosis/necrosis. Previous studies have indicated that endoplasmicreticulum (ER) stress is involved in the cell death process. Thisresults in secondary activation of the innate immune response involvingup-regulation of inflammatory cytokines with activation of NK, NKT cellsand neutrophils, which significantly contributes to hepatotoxicity andmortality (3, 4). APAP overdose as a result of suicidal or unintentionalingestion is a critical issue [6, 7], and the incidence of APAP-inducedliver injury has been increasing in recent years [8]. In the UnitedStates [2], the United Kingdom, and other countries [3-5]. Thefirst-line therapy for APAP overdose is treatment with oral N-acetylcysteine, which is a glutathione precursor; however, this hasdose-related adverse effects and limited therapeutic efficacy [9].Therefore, preeminent need to explore alternative therapeutic approachesagainst APAP-induced hepatotoxicity. The present invention an optimizedacetaminophen phenylbutyrate ester composition aimed to provide asolution for above mentioned problems.

BRIEF SUMMARY OF THE INVENTION

The present invention to provide an optimized acetaminophenphenylbutyrate ester preparation and composition suitable for oral,injectable administration which prevents or reduces hepatotoxicityproblems and treat, Pain, fever, inflammation, a composition for thetreatment of ischemic injury or nerve damage, pharmaceuticallyacceptable salt or composition comprising a solvate of the foregoing.

It is a further aim of the present invention to provide a method ofmanufacturing or synthesis of drug-Phenylbutyrate conjugates, orpharmaceutically acceptable salt and an improved phenyl butyrate drugconjugate composition suitable for human or animal use thereof. It is ayet further aim of the present invention to provide a method of use ofan improved acetaminophen ester composition for oral, injectableadministration.

One aspect of the present invention provides a compound, or apharmaceutically acceptable salt thereof or solvate of the foregoingcomprising an acetaminophen moiety and a PBA moiety.

In some embodiments, the present invention comprises a compoundcomprising an acetaminophen moiety, or other drugs moieties and a PBAmoiety.

All publications cited herein, patents, patent applications anddisclosure other references in their entirety are incorporated herein byreference. German Patent Application Publication No. 4327462 Pat

BRIEF DESCRIPTION

Synthetic schemes for target compounds: compound can be prepared usingdifferent coupling ages is and reagents and acid, bases with protectinggroups commonly used in organic synthesis.

Synthetic Scheme for Formula-I:

In above R can be ═OH, or O-Alkyl, O-Aryl or O-Heteroaryl, anyprotecting group

-   -   Drugs molecules reported in “Hepatotoxicity by

X═OH, NH, SH, SO₂ or NH₂

-   -   Drugs: The Most Common Implicated Agents”

Drug=Hepatotoxicity induceable drugs.

-   -   International Journal of Molecular Sciences reported by Einar S.        Björnsson et. al., Int. J. Mol. Sci. 2016, 17, 224;        doi:10.3390/ijms17020224. And all the references sited over        there.

Synthetic Scheme for Formula-II:

-   -   In Above R₁=Me, or Ethyl and R═H, or Protecting groups    -   R₂═H, Me, Ethyl, Heteroaryl, Protecting groups    -   Reagent can be coupling agents reported in organic synthesis;        such as DCC, HATU, HOBT, DIPEA, TEA, etc.,

General Synthetic Procedure:

Drug (1 mol) was dissolved in 50 ml Solvent, 4-Phenylbutyric acid oracid chloride or ester (1.1 mol) was added and base (0.2 mol) was addeddrop wise into the reaction. Coupling agent (0.2 mol) was added portionwise reaction mixture was stirred at room temperature for 5-24 hrs, thereaction was monitored using TLC, after completion of starting materialReaction mixture was poured in to 100 ml water and extracted withorganic solvent 100 ml twice, the combined organic layer was washed withwater twice and dried over sodium sulphate and solvent removed underreduced pressure, crude product was purified by column chromatography toobtain final product formula I and II.

Procedure for Formula II:

N-(4-hydroxyphenyl)acetamide (1.5 gm, 0.99 mol) was dissolved in 100 mlDMF, 4-Phenylbutyric acid (16.2 gm, 0.99 mol) was added and base (Triethyl amine 20.8 ml, 0.148 mol) was added drop wise into the reaction.Coupling agent (HATU 45 gm, 0.1188 mol) was added portion wise reactionmixture was stirred at room temperature for 18 hr. the reaction wasmonitored using TLC, after completion of starting material Reactionmixture was poured in to 200 ml water and extracted with Ethyl acetate100 ml×3 times, the combined organic layer was washed with water 200ml×2 times and dried over sodium sulphate and solvent removed underreduced pressure, crude product was purified, final product 22 gm ofN-(4-phenylbutanoyl)-N-(4-hydroxyphenyl)acetanyide was obtained.

Formulation Preparation:

The formulations were prepared as follows: Acetaminophen phenylbutyrateconjugate was added to a mixture of 5%-10% Polysorbate 80 and waterwhile stirring and mixing for 30 minutes to form stable micelles. Thevolume was then increased to 10 ml with WFI water to prepare liquidconcentrate formulations. The conjugate concentrates can be diluted withany quantity of commonly used intravenous infusion solutions. The liquidconcentrate can be taken as oral liquid.

Amber glass bottles were filled with the above formulations of 10 mLconcentrate, with a rubber stopper and flip-off seal and subjected tostability studies under the following conditions:

-   In some embodiments polysorbate 80 percentage in formulations can be    between 0.5% to 25%.-   In some embodiments drug (APPB) concentration in formulations can be    20 mg/ml to 150 mg/ml.

ICH accelerated conditions at 40° C.±2° C./75% RH±5% RH; and

ICH room temperature conditions at 25° C.±2° C./60% RH±5% RH

Samples were analyzed to measure the acetaminophen phenylbutyrateconjugate assay, impurities. Also, physical stability of the inventedformulation example physical appearance and pH drift was recorded. Thestability of the concentrate is stable no assay drop or precipitationobserved, and no impurities formation observed.

In Vivo Safety and Dose Ranging Study:

A safety study was conducted in healthy 18 (male and female (M&F)Sprague Dawley (SD) rats, animals were divided into 3 groups 6 rats(3(M) & 3(F)) in each group, Group-A low dose 500 mg/kg/per day andGroup-B medium dose 1000 mg/kg/per day and Group-C high dose 1500mg/kg/per day for 5 days doses were given orally. No treatment relatedclinical signs were noticed throughout treatment duration and dailyactivities such as casting food and drinking water and moving. All groupanimals were survived no anomalies observed during and after treatmentperiod animal were under observation for one week. This study clearlydemonstrates this new conjugate (APPB) is safe for prolonged use athigher doses.

In Vivo Comparative Drug Induced Hepatotoxicity Challenge Study:

An in-vivo study was performed in healthy SD rats to evaluate drugefficacy. A single dose and multi dose parallel study was conducted toevaluate the drug induced hepatotoxicity by measuring plasma AST and ALTlevels. Following acclimation, overnight-fasted 6-week-old 12 SD-ratswere randomly divided into two groups, 6 animals in each group (3M&3F),Group-A Test compound Acetaminophen phenylbutyrate (APPB) and Group-Breference compound Acetaminophen (AAP). Group-A animals were treatedwith Test compound (APPB) 800 mg/kg body weight (BW) intraperitoneal(IP) injection of APPB conjugate given and 400 mg/kg body weight (BW)reference compound (AAP) IP injection Oven to Group-B animals, andplacebo IP injection given to 6 animals (3M&3F) in group-C.

randomly divided into 3 groups were given an intraperitoneal (i.p.)injection of APAP [450 mg/kg body weight (BW)]. Some of these mice werethen repeatedly injected with PBA (120 mg/kg BW, i.p.) every 3 h from0.5-3 to 12 h after

The drug induced hepatotoxicity challenge, In-life observations includedcage side clinical sign observations, body weight and ALT estimationprior to start of treatment on day −1 and then reference group mice wereserially euthanized by blood withdrawal after 1, 3, 6, and 12 h, afterAPAP injection, whereas the APPB-treated mice were euthanized by bloodwithdrawal after 1, 3, 6, and 12 h.

The levels of serum aspartate aminotransferase (AST) and alanineaminotransferase (ALT) activity were measured colorimetrically

In Vivo Comparative Pain-Relieving Model Study:

12 Rabbits (M&F) (formaldehyde pain model 6 hr) were divided into 3groups 4 rabbits in each group, Group-A Test (Acetaminophenphenylbutyrate) and Group-B reference (Acetaminophen) and Group-CControl (placebo). Group-A 4 rabbits (2M & 2F) were treated with Test300 mg/kg orally given and 150 mg/kg; reference compound given toGroup-B 4 rabbits (2M & 2F) and placebo given to group-C after 15 mineach rat was dosed with 2.5% formalin in saline solution 0.1 ml wasinjected into middle of paw, and monitor for number of licking orscratching's happened with each animal in all groups and compared eachgroup with other in the below table.

FIG. x. Amplitude of rubbing activity at formalin injected site for 60min post dose of formalin in test, reference and placebo. Amplitude ofrubbing Treatment Rabbit No. of activity (Group. No.) Id rubbings(seconds) Sum Mean SD Group-1 1M 3 11.84 36.19 3.62 9.9 Test 2M 2 6.69Conjugate drug 3F  1 5.02 APPB 4F  4 12.64 Group-2 5M 1 3.1 16.7 2.786.0 Reference 6M 2 6.7 Acetaminophen 7F  1 2.6 8F  2 4.3 Group-3 9M 29.42 57.42 5.03 11.41 Control 10M  5 21.9 Placebo 11F   2 9.5 12F   319.6 Note: The data was analysed statistically using unpaired t-test forcomparison of test formulation 1 and reference formulation 2 withformulation 3 (placebo). The level of significance was set at p < 0.05.Data was expressed as Mean ± SD.

In Vivo Comparative Antipyretic Effect Study:

The in vivo pharmacological study conducted the anti-pyretic activity ofacetaminophen Phenylbutyrate (APPB) formulations in SD rats againstyeast induced pyrexia 12 rats (M&F) (Baker's yeast induced antipyreticmodel 24 hr) After measuring basal T_(R) of the animals, they wereinjected with a pyrogenic dose of Baker's yeast (0.135 g/kg, i.p.).T_(R) changes were recorded every hour up to 4 h. Animals that showed anincrease of not less than 0.5° C. in rectal temperature were selectedfor the experiment. Animals were randomly divided into three groups 4rats in each group, Group-A Test (Acetaminophen phenylbutyrate) andGroup-B reference (Acetaminophen) and Group-C Control (placebo). Group-A4 rats (2M & 2F) were treated with Test 300 mg/kg orally given and 150mg/kg reference compound given to Group-B 4 rats (2M & 2F) and placebogiven to group-C. From the time baker yeast injection body temperaturewill rise keep monitor every 30-60 min once and record. And aftertreatment (time after treatment with test, reference and placebo) thebody temperature was monitored rectally, for next 6 hr (0, 0.5, 1.0,1.5, 2, 3, 4, and 6) using digital thermometer which can capture 0.1 Fbody temp ad recorded. The collected data was presented below table.

TABLE X Effect of APPB-conjugate prepared formulation on yeast- inducedpyrexia at various time intervals in SD rats Group 3 hours 6 hours 9hours Test Group-A 1.21 ± 0.13 1.48 ± 0.08 1.83 ± 0.06 Reference Group-B0.83 ± 0.08 1.31 ± 0.23 1.91 ± 0.14 Placebo Group-C 2.36 ± 0.06 2.57 ±0.11 2.93 ± 0.08 Data was expressed as Mean ± SD.

shows data related to the effect of APPB-conjugate on yeast-inducedpyrexia at different time intervals. Yeast injection in experimentalanimals caused significant rise in body temperature at the various timeintervals as recorded rectally with the help of a tele-thermometer.Reference compound, a well-established antipyretic drug attenuated therise in temperature to a significant extent at all time intervals. TheAPPB-conjugate significantly attenuated the rise in temperature 3 hafter yeast injection After 6 and 9 h of yeast injection also theAPPB-conjugate attenuated the raise in temperature in a highlysignificant manner in comparison to placebo control group.

As used herein, “pharmaceutically acceptable” means biologically or nomaterial be undesirable in other respects, for example, the material,significant undesirable biological histological effect without the bringany, or without interacting in a deleterious manner with any of theother components of it composition included, can be incorporated into apharmaceutical composition administered to an individual (e.g., at thetime of manufacture or administration). As used herein, the term“pharmaceutically acceptable carrier”, for example, an individual (e.g.,human) solvents known to those skilled in the art suitable foradministration to, stabilizing agents, pH adjusting agents, tonicityadjusting agents (tonicity modifier), adjuvants, binders, means such asa diluent. Combinations of two or more carriers are also contemplated inthe present invention. As described herein, the componentpharmaceutically acceptable carrier (s) and any additional, adapted foruse in the intended route of administration of the particular dosageform (e.g., oral, parenteral) there should be sex. Such compatibility,particularly in view of the teachings provided herein will be readilyrecognized by those skilled in the art. Pharmaceutically acceptablecarriers or excipients, contained in the pointer (Inactive IngredientGuide) related to An effective amount” means that the composition to beadministered, the condition being treated/prophylactically (e.g., thetype of pain), the severity of the condition being treated or prevented,the individual's age, body size, body weight and relative health ofadministration route and form, the attending physician or veterinariandetermined (if applicable) and in view of the teachings describedherein, can vary by other factors that are well understood by thoseskilled in the art. An effective amount, for example, can be assessed byusing one or more clinical, physiological, biochemical, histological,data from electrophysiological and/or behavioral evaluations.

In some embodiments, the individual is bovine, horse, cat, rabbit, dog,including rodents or primates, but not limited to, a mammal. In someembodiments, the mammal is a primate. In some embodiments, the primateis a human In some embodiments, the individual is a human, includingadults, children, infants and premature infants. In some embodiments,the individual is a non-mammal. In some variations, the primate is anon-human primates such as chimpanzees and other apes and monkeyspecies. In some embodiments, the mammal is a cow, horse, sheep, cattle,such as goats and swine; is rat, etc. laboratory animals includingrodents such as mice and guinea pigs; rabbits, pets such as dogs andcats . In some embodiments, the individual including birds, is anon-mammal, including but not limited to. The term “individual” does notdenote a particular age or gender. inactive ingredients created bysatisfying the required standards of toxicological and manufacturingtesting, and/or the U.S. Food and Drug Administration it is preferredthat the.

1. (canceled)
 2. A compound of the formula (F-II): or a pharmaceuticallyacceptable salt thereof and its use to treat or cure liver diseases orof liver necrosis, and nonalcoholic steatohepatitis (NASH), and treatPain, fever, cancer, inflammation, mentioned in above in individuals,human or animal, wherein R1 is methyl.
 3. A formulation comprising acompound of claim 2 and a pharmaceutically acceptable carrier or carriercomprises composition a solvate of the foregoing.
 4. A method oftreating a disease or condition selected from the group treat, reduce orcure liver and disease consisting of pain, fever, inflammation, ischemicinjury, and neuronal injury, comprising administering to an individualin need thereof an effective amount of a compound according to claim 1.5. The method of claim 6, wherein the compound is administered orally orparenterally.
 6. (canceled)
 7. (canceled)
 8. A method of treating adisease or condition selected from the group treat, reduce or cure liverand disease consisting of pain, fever, inflammation, ischemic injury,and neuronal injury, comprising administering to an individual in needthereof an effective amount of a compound according to claim
 2. 9. A kitfor the treatment or prevention of a disease or condition selected fromthe group consisting of pain, fever, inflammation, ischemic injury, andneuronal injury, comprising a compound of claim 2, or a pharmaceuticallyacceptable salt thereof and instructions for us.